The long term objective of this grant is to advance fundamental knowledge of the mechanism of protein hydrogen exchange, a unique and versatile probe of protein dynamic structure. Hydrogen exchange in proteins occurs either by internal motions of the folded state, or by global cooperative unfolding, and we call this the two-process model of exchange. To advance understanding of each process, we characterized relationships between hydrogen exchange rates and average (crystal) structure of the folded state, as well as relationships between hydrogen exchange rates and global unfolding. Toward this end, the work funded by this grant since the last competitive renewal produced a significant body of data on dynamics, folding thermodynamics and crystal structure for 8 variants of the small model protein, bovine pancreatic trypsin inhibitor (BPTI). The research plan in this proposal is twofold; i) to bring the data base we have built to bear on further important questions of mechanism of hydrogen exchange in proteins and of the relationship of dynamics measured by hydrogen exchange to protein average structure (crystal or NMR) and to protein folding, and ii) to test our hypothesis relating protein dynamic structure to protein folding, namely, that the native state slow exchange core is the folding core. Specific aims include the following. a) Exchange rates of surface NHs in BPTI mutants will be determined. b) Double mutant combinations of our single-site mutants will be constructed in order to examine BPTI denaturation at low pH. c) Highly accurate thermodynamics for the cooperative folding transition of single site BPTI mutants will be obtained in collaboration with P. Privalov. d) Pulsed exchange experiments on BPTI mutants will be carried out. e) NMR and hydrogen exchange experiments will be carried out on BPT1 analog [14-38]Abu which retains the 14-38 disulfide bond but has the other four cysteines replaced by alpha-amino butyric acid. We will characterize the hydrogen exchange properties, folding thermodynamics and NMR dynamic structure of [14-18]Abu at pH 4.5-6 and 3-10 degree C, conditions under which it is an ensemble of partially folded structures, and a model for early intermediates in BPTI folding. Other NMR experiments are planned for variants of [14-38]Abu under partially folding conditions, and for [14-38]Abu at pH less then 2 and 5 degree C, conditions under which it forms an acid state (A state). f) NMR structures and hydrogen exchange of two analogs of reduced BPTI, models for the unfolded state of the protein, will be characterized. Preliminary evidence strongly suggest hydrophobic clustering and non-random, possibly non-local, NMR-detected structure in reduced BPTI.